The invention relates to the manufacture of media that can be used as support in various adsorption processes in order to enrich substances from various liquids, in cell culture, in synthesis taking place on supports and in other fields related to these fields.
The term media refers to the matrix plus the pore volume.
The stationary phase in liquid chromatography is composed of a matrix and is mostly in the form of porous or nonporous particles packed to a bed or in the form of a porous monolith. The matrix should be rigid enough to allow high flow rates without being compressed or broken, for instance when performing separations in columns packed with particles of small diameters. The matrix should also be chemically and physically inert and withstand extreme pH conditions.
In reversed phase chromatography (RPC), particularly for peptide and protein separation, silica gels have for a long time been the preferred stationary phase. These matrices are rigid enough to withstand high flow rates. However, silica gels are not stable above pH 8.0. During recent years there has been a growing interest in synthetic polymeric materials for use in reversed phase high performance liquid chromatography (HPLC), in particular resins based on polymerisation of mono-, di- etc vinyl compounds with particular emphasis of poly vinyl hydrocarbons such as copolymers between styrene and divinylbenzene). These media are often stable with eluents having pH within the range 1-14 and often give excellent separations. The amount of swelling may differ widely depending on fraction of cross-linker to monomer and the liquid they are contacted with. They normally comprise some free unreacted residual vinyl groups (again depending on fraction of cross-linker to monomer) and are not compressed by the pressures normally applied in HPLC.
Polymerisation of vinyl compounds in order to accomplish cross-linked polymerisates/copolymerisates may take place stepwise with a first step resulting in linear non-cross-linked polymers and then in a second step at an increased temperature cross-linking takes place (curing). Often nothing is removed or added between these two steps meaning that for instance significant amounts of initiators and monomers or oligomers with unreacted vinyl groups from the first step remains and are used in the second step. The polymerisate from the first step is characterised by being soft and possible to shape by heating. The second step is often called curing and thus results in a cross-linked copolymerisate. The concept of curing is often utilised in polymerisations, see for instance U.S. Pat. No. 4,977,222; EP 286071 (step e); WPI accession number JP 81-82583D (=JP 56122814) and WPI accession number 81-75832D (DD149672).
WPI accession number 91-257239 (=JP 03168204) describes a cross-linked product from which porogens, monomers, initiators etc are removed in a heating step.
Technical Problem With Prior Art Matrices Based on Polymerised Vinyl Compounds
The rigidity of the media is a key issue regarding its ability to withstand high pressures during packing of columns and separations. The packing of media in the form of particles are performed by injecting a suspension of the material into the column with a pressure of several hundred bar. During high performance liquid chromatography (HPLC) the pressure drop over the column can be as high as 200-400 bar.
In order to fulfil these needs a highly cross linked matrix is required. Further, the percentage of polymer matrix in the particle must not be too low. Unfortunately the higher percentage matrix in the media the more the media will swell in an organic solvent.
The ability of media based on vinyl compounds to swell in the presence of organic solvents is one of their major disadvantages when used as the stationary phase in applications requiring flow through the stationary phase, such as in chromatography utilising increased pressure. This becomes particularly troublesome in processes requiring changes in solvents, for instance when solvent gradients are used, such as in reversed phase chromatography (RPC).
A first objective is to provide methods for producing media that are improved concerning the above-mentioned drawbacks.
A second objective is to provide media in which these drawbacks have been reduced.
We have now discovered a method for reducing the swelling of vinyl based media without having to change the media composition. Accordingly, one aspect of the invention is a method of manufacturing a media for use as support in the technical field of the invention, preferably in processes requiring contact with organic solvents and optionally with application of liquid pressure. The starting media is a cross linked polymerisate of vinyl compounds, preferably a copolymerisate between a monovinyl compound and a compound exhibiting two or more, preferably two vinyl groups, so that the copolymerisate (media) will exhibit residual vinyl groups. This means that the copolymerisate used in the inventive process is ready-made in the sense that essentially all residual monomers and/or initiators remaining after polymerisation and/or porogens, if present during polymerisation, should have been removed. The characterising feature of the method is that the media (support) is subjected to a heating step, which preferably is carried out at reduced access of oxygen.
The temperature during the heating step should be such that the swellability is decreased. The actual temperature may vary depending on polymer and heating time. A prolonged heating can, for instance, compensate for a lower temperature. It is important not to heat to the extent that a decomposition starts that is harmful to the desired properties of the media. The heating temperature and time thus shall be such that carbonisation does not take place. Normally the temperature is below 500xc2x0 C. and above 75xc2x0 C. A representative interval is 120-350xc2x0 C. with preference in many cases for the interval 180-300xc2x0 C. These rules particularly apply for matrices based on unsaturated hydrocarbons (vinyl hydrocarbons), such as styrene divinyl benzene.
Reduced access of oxygen means that the heating step may be run under reduced atmospheric pressure or under an inert gas, for instance argon or nitrogen. The main point is that the oxygen pressure is reduced relative to partial oxygen pressure in air at atmospheric pressure. By this term is also contemplated that the matrix is protected from contact with the atmosphere due to being surrounded by a liquid, for instance a hydrocarbon liquid. Particles may for instance be suspended and a monolithic matrix soaked in an inert liquid, such as a hydrocarbon liquid. Often it is advantageous to combine an inert liquid with the application of an inert atmosphere above the liquid, for instance argon or nitrogen.
It has been found that the above-mentioned conditions for the heat treatment may result in matrices exhibiting a reduced back pressure in a packed bed.
It has also been found that it is difficult to prevent oxidation during the heat treatment and that formed oxidised groups have a negative impact on the performance if the media is used in chromatography. It has therefore been found beneficial to post-treat the matrices under conditions providing reduction of oxidised groups formed during the heat treatment. Illustrative reductive agents are for instance those that reduce carbonyl groups, such as in aldehydes and ketones, to alcohol groups or to hydrocarbons. Specifically can be mentioned sodium boro hydride and lithium aluminium hydride. Similar conditions as used elsewhere apply to the respective reductive agent.
The vinyl compound is a compound that exhibits a polymerizable carbon-carbon double bond, such as in vinyl hydrocarbons (vinyl aryls and alkenes), vinyl ethers, acrylates and methacrylates, acrylamides and methacrylamides etc. The term vinyl compound encompasses mono-, di-, tri- etc vinyl compounds. A specifically preferred group of vinyl compounds is the vinyl hydrocarbons, in particular in form of vinyl aryls, for instance various isomers and substituted forms of styrenes and divinyl benzenes. Other examples of vinyl hydrocarbons are buten and 1,3-butadiene. The vinyl compound may contain various substitutes, such as C1-26, with particular emphasis of C1-16, alkyl groups and functional groups that are compatible with the copolymerisation reaction intended. This has typically been the case for various vinyl benzenes. Copolymerisates of vinyl compounds, in particular between styrenes and divinyl benzenes, have for quite a long time been used as commercially available supports in the field of the invention.
A general property of the media to be used in the present invention is that they swell in contact with an organic solvent, such as methanol, acetonitrile, tetrahydrofuran and other organic solvents used in liquid chromatography. The swelling can be studied, for instance, by measuring the difference of the packed bed volume when going from water to any of the organic liquids just mentioned. Since there always is a desire to minimise this drawback, the invention in principle provides advantages even for media giving an infinitesimal swelling. This latter may be illustrated by the fact that the invention may provide advantages for all media having an increased swelling  greater than 0.5% (by volume) when going from water to any of the solvents given above (measured as the volume of a packed bed or a monolithic bed). An better indication of decreased swelling is the lowering in pressure drop found over a bed comprising the matrix in an organic liquid before and after the matrix has been heated in accordance with the invention (packing pressure, applied flow etc all being the same in the comparison).
The number of residual vinyl groups in the polymerisate should typically be within the interval 0.1-5 mmole/g before the thermal treatment start and will typically diminish 1-50% during the heat treatment (measured by bromination). The decrease may differ depending on the specific conditions and methods applied. For instance, treatment at reduced pressure may give a reduction that is lower than treatment in a liquid.
The matrices may be in the form of a porous monolith or in the form of particles that may be porous or non-porous. Porosity may be measured in different ways: surface area per g matrix, pore volume per g matrix and mean pore diameter. The total surface area can be measured by nitrogen adsorption (BET method). The volume of pores with diameters larger than 25 xc3x85 can be measured by mercury porosimetry, and smaller pores can be measured by nitrogen desorption.
The selection of matrices having a certain porosity is primarily dependent on the intended use of the media.
For chromatography and other separation purposes it depends on type of chromatography, what to be chromatographed etc. It will also depend on if only diffusive mass transport or diffusive and convective mass transport through the matrix is desired. Diffusive mass transport then normally being satisfied with pore sizes below 1 xcexcm and convective mass transport taking place, mainly in pores larger than 1 xcexcm (flow through pores). For matrices in the form of particles the ratio between pore size and particle diameter normally should be in the range of 0.05-0.5 for flow through pores. Thus the pore size diameter may range from a few xc3x85, such as 50 xc3x85, up to very large values, such as 50 xcexcm. The general condition that the pore diameter always is significantly less than the particle size applies.
The porosity in this field of use typically is: a surface area in the interval 50-1000 m2/g matrix, a pore volume in the interval 0,5 xe2x88x923 ml/g matrix and a mean pore diameter that may vary from 100-2000 xc3x85.
For porous monoliths the corresponding porosity intervals may be broader, for instance 50 xc3x85-50 xcexcm for pore size diameters.
For cell culture the media may be porous or nonporous. In many cases there will be advantages in case the pores are of such a size and form that they may harbour the cells to be cultured.
As demonstrated in the experimental part, the heat treatment does not need to cause any changes in the porosity.
For particulate matrices the particle size normally is within the range 1-500 xcexcm. Particulate matrices may be monosized (monodispersed) or polysized (polydispersed). By a monosized matrix is typically meant a population of matrix particles having more than 95% of the particles with sizes within mean diameter xc2x15% of the particles of the population.
After the heat treatment the matrix may be derivatized as well known in the art. For instance by setting the proper hydrophilic/hydrophobic balance on the matrix and/our adding specific binding groups, for instance selected among affinity groups, such as biospecific affinity groups, ion exchange groups (cation or anion exchange groups), amphoteric groups, hydrophobic groups, chelating groups, groups allowing xcfx80xe2x80x94xcfx80 interactions, and groups allowing covalent chromatography. Since one of the largest advantages with the method according the invention is to prepare matrices that can be used together with organic solvents, derivatization often means that a certain degree of hydrophobicity in most cases should be retained. The matrices obtained according to the invention is primarily intended to be used in chromatography utilisutilising organic solvents, with particular emphasis of high pressure liquid chromatography (HPLC). In HPLC the applied pressure is often in the interval 30-300 bar, such as 50-200 bar. One can envisage that the upper limit may go up to 500 bar or even higher. Popular organic solvents are acetonitrile, methanol and tetrahydrofuran. This, however, does not exclude that the matrices also can be used in the connection with aqueous solvents or as support in quite other fields, such as carrier/support in solid phase synthesis, for instance of oligopolypeptides and oligonucleotides, as carrier/support in cell culture etc. The matrices may also be used in other processes involving adsorption from a liquid in which the support functions as the adsorbent (for instance batch-wise processes based on the various affinity groups discussed above).